In diverse research fields and industrial fields, conventionally, an approach for simply and rapidly obtaining the sequence information of an oligonucleotide or a polynucleotide (nucleic acid) as DNA or RNA is highly needed.
Furthermore, a research and technical field called RNAi (RNA interference) starting from the discovery that a relatively short double-stranded RNA exerts a strong gene silence effect in vivo has been spread rapidly in recent years. In the field of RNAi, an approach for obtaining for example the sequence information of the double-stranded RNA as a relatively short oligonucleotide simply and rapidly has an extremely important meaning.
Conventionally, meanwhile, methods for modifying or chemically converting nucleic acid bases in a manner selective to base species have been known, like for example the well-known Maxam-Gilbert method.
Additionally, the following patent reference 1 discloses a technique for labeling the end of a reducing sugar comprising allowing for example 2-aminopyridine together with a reducing agent to react with the end thereof.
[Reference 1] JP-A-2007-038130
By the Maxam-Gilbert method, however, only a single nucleic acid base in a subject nucleic acid chain is released; subsequently, the chain is cleaved starting from the site under basic conditions. By the method, therefore, not all of the bases of one nucleic acid base species in the subject nucleic acid chain are substituted and labeled.
Further, the patent reference 1 discloses one type of sugar chain-labeling techniques for simply labeling the end of a reducing sugar through a reducing amination. As an elemental technique, the patent reference 1 has some relation with the object of the invention but essentially, the patent reference 1 does not have much relation with the invention.
Alternatively, the reaction of a poly- or oligodeoxyribonucleotide including adenine, guanine, cytosine and thymine or a poly- or oligoribonucleotide including adenine, guanine, cytosine and uracil with chloroacetaldehyde to modify the adenine and the cytosine therein has been known (the following reference 2). Such modified nucleosides have a property of emitting fluorescence and are converted to monomers for use with an automatic oligonucleotide synthetic apparatus; and then, the monomers are incorporated into an oligonucleotide with the synthetic apparatus for use as a fluorescent nucleic acid probe and the like (the following reference 3).
[Reference 2] Biochemistry, vol. 11, No. 19, 3499-3506 (1972)
[Reference 3] Biochemistry, vol. 26, No. 19, 5626-5635 (1987)
Although the method of the reference 2 is a simple and secure method, the method is used just for modifying only adenine and cytosine. By the method alone, thus, all bases constituting DNA or RNA cannot be discriminated.
The group of the present inventors has made examinations about the analysis of DNA and RNA sequences with an electron microscope by labeling bases constituting DNA and RNA. A method for labeling individual bases constituting DNA and RNA in a simple manner has been demanded.